Purification and Characterization of a New Thermostable Laccase from Bacillus licheniformis O12 Using One-Step Affinity Chromatography and Its Potential for Decolorization

Öz

Laccases are copper-containing enzymes that can oxidize a wide variety of substrates. Thanks to this feature of laccase, some dyes that cause environmental pollution can be decolorized. Some bacteria, such as Bacillus licheniformis, naturally produce the enzyme laccase. A new affinity column was tested in this study. For this purpose, the extracellular laccase sepharose 4B-L-tyrosine-ρ-aminobenzoic acid produced by bacteria grown in suitable media was isolated by affinity chromatography method. Its purity was checked by SDS-PAGE method. The decolorization effect of some dyestable in textile wastewater of laccase isolated from B. lichenisformis O12 by affinity column was investigated. No mediator was used in this procedure. .As a result, laccase was purified 4.82-fold purification with a yield of 38.3% respectively, The molecular weight of the purified enzyme was determined as ~70 kDa by the SDS-PAGE method. The enzyme showed optimum activity at pH 4.0 and temperature 92°C. The enzyme was found to retain 100% activity even after 12 hours of incubation at 60°C and 92°C. The kinetic parameters were determined with laccase substrates such as ABTS, 2,6-DMP, and guaiacol. The purified laccase was decolorized with varied efficiencies such as 35% of Reactive black, 31% of Acid black 1, 28% of Methylene blue, and 15% of Acid red 27 without the use of any redox mediators. These properties of B. licheniformis O12 laccase enzyme make it a potential candidate enzyme for use in various biotechnological and industrial applications.

Anahtar Kelimeler

• Bacillus licheniformis
• Laccase
• Affinity chromatography
• Characterization
• Decolorization
• Purification

Bacillus licheniformis O12'den Yeni Bir Termostabil Lakkazın Tek Adım Afinite Kromatografisi Kullanılarak Saflaştırılması, Karakterizasyonu ve Renk Giderme Potansiyeli

Öz

Lakkazlar çok çeşitli substratları oksitleyebilen bakır içeren enzimlerdir. Lakkazın bu özelliği sayesinde çevre kirliliğine neden olan bazı boyarmaddelerin renk giderimi yapılabilmektedir. Bacillus licheniformis gibi bazı bakteriler doğal olarak lakkaz enzimini üretir. Çalışmada lakkaz saflaştırması için yeni bir afinite kolonu test edilmiştir. Bu amaçla uygun ortamda yetiştirilen bakterilerin ürettiği hücre dışı lakkaz sefaroz 4B-L-tirozin-ρ-aminobenzoik asit afinite kromatografisi yöntemiyle izole edildi. Saflığı SDS-PAGE yöntemiyle kontrol edildi. B. lichenisformis O12'den afinite kolonu ile izole edilen lakkazın tekstil atık suyundaki bazı boyarmaddelerin renk giderici etkisi araştırıldı. Bu prosedürde herhangi bir mediyatör kullanılmadı. Sonuç olarak lakkaz %38,3 verimle 4,82 kat saflaştırıldı. Saflaştırılan enzimin moleküler ağırlığı SDS-PAGE yöntemiyle ~70 kDa olarak belirlendi. Enzim, pH 4.0'da ve 92°C sıcaklıkta optimum aktivite gösterdi. Enzimin, 60°C ve 92°C'de 12 saatlik inkübasyondan sonra bile %100 aktivitesini koruduğu görüldü. Kinetik parametreler ABTS, 2,6-DMP ve guaiacol gibi lakkaz substratları ile belirlendi. Ortama sadece izole edilen lakkaz eklenerek, herhangi bir redoks aracısı kullanılmadan %35 Reaktif siyah, %31 Asit siyahı 1, %28 Metilen mavisi ve %15 Asit kırmızısı 27 boyar maddelerinde renk giderimi elde edildi. B. licheniformis O12 lakkaz enziminin bu özellikleri, onu çeşitli biyoteknolojik ve endüstriyel uygulamalarda kullanım için potansiyel bir aday enzim haline getirmektedir.

Anahtar Kelimeler

• Bacillus licheniformis
• Lakkaz
• Afinite Kromatografisi
• Karakterizasyon
• Renk giderimi
• Saflaştırma

Kaynakça

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